Detection of Oligosaccharides Labeled with Cyanine Dyes Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Abstract

The sensitivity of oligosaccharides in mass spectrometry lags far behind that of peptides. This is a critical factor in realizing the high-throughput analysis of posttranslational modifications in proteomics. We here described that hydrazide derivatives of cyanine dyes (Cy3, Cy5) with a positive charge made excellent labeling reagents for the detection of oligosaccharides by matrix-assisted laser desorption/ionization mass spectrometry. Cy3-labeled standard N-glycan could be detected at 200 amol on the MALDI target plate in reflectron mode without any purification procedures after the labeling reaction, which may meet the level of sensitivity required in proteome research. Despite the general recognition that the production of signals of oligosaccharides under MALDI conditions would be highly dependent on the matrix, most of the known N-glycans from chicken ovalbumin could be detected upon Cye derivatization nearly independent of the kind of matrix tested (e.g., nor-harman, 2,5-dihydroxybenzoic acid and alpha-cyano-4-hydroxycinnamic acid) without spoiling the signal strength. Postsource decay afforded simple spectra mainly consisting of Y-type fragment ions, thus simplifying the sequence analysis. In-source decay afforded a similar fragmentation pattern only when acidic matrixes were used. In addition, this derivatization technique was successfully applied to the profiling of N-glycans of gel-separated glycoproteins.

Identification and characterization of sulfated glycoproteins from small cell lung carcinoma cells assisted by management of molecular charges

“…Charge control by chemical modification is a key concept required for this achievement. In studies using mass spectrometry, many reports have described improvements in detection and/or fragmentation by attaching an additional charge to the analyte [18,19,37]. In this study, neutralization and addition of molecular charge were utilized at multiple stages in the analytical procedure.…”

“…However, artificially induced charges can sometimes be introduced for analytical purposes in electrophoresis and mass spectrometry analysis [17][18][19]. Previously, we developed a novel procedure to enrich for sulfated glycoproteins, termed the sulfate emerging (SE) method [20], which enhanced the negative charge of the sulfate group by managing the net charge of the peptide.…”

2-Aminobenzamide and 2-Aminobenzoic Acid as New MALDI Matrices Inducing Radical Mediated In-Source Decay of Peptides and Proteins

“…M ALDI in-source decay (or MALDI ISD) has been observed for various kinds of analytes such as peptides and proteins [1][2][3][4][5][6], carbohydrates [7][8][9][10][11][12] oligonucleotides [13], and is currently used for top-down protein sequencing [14]. Concerning peptides and proteins, two main activation modes, thermal and radical-mediated, were described in literature to explain the in-source fragmentation pathways.…”

Quantitative Glycomics

“…Other reported hydrazide labeling reagents for glycomics include Biotinyl-L-3-(2-naphthyl)-alanine hydrazide (Walker, Lilley, Enamorado, Comins, & Muddiman, 2011; Walker, Papas, Comins, & Muddiman, 2010), keratan sulfates (Y. Zhang, Cai, Zhao, & Luo, 2008), cyanine dyes (Kameyama, Kaneda, Yamanaka, Yoshimine, Narimatsu, & Shinohara, 2004) and 4-phenyl pyridine(Bereman, Comins, & Muddiman, 2010). …”